ANTRES Informe resumido

Development and validation of molecular tools for the genetic analysis of resistance mechanisms and genotypes

- Detection of resistance genes and integrons. Molecular methods, based on DNA hybridization or PCR and direct sequencing, for detection of the following resistance genes were developed and validated: -lactamases resistance genes (blaTEM, blaSHV, blaCTX-M, blaPER and various blaOXA), tetracycline resistance genes (tet(A), tet(B), tet(C) and tet(D)), phenicols resistance genes (catI and cmlA), trimethoprim resistance genes (dfrA1, dfrA5, dfrA7, dfrA8, dfrA12, dfrA13, dfrA14 and dfrA17), sulphonamides resistance genes (sul1, sul2 and sul3). As in gram negatives the aminoglycoside resistance determinants are known to be usually integron-associated, a PCR mapping approach was developed, suitable for identification of integron-borne aminoglycoside resistance genes. The method is based on PCR amplification of integron cassette arrays using primers designed on conserved integron regions (class 1 and class 2 integrons), possibly in combination with primers specific for the various aminoglycoside resistance genes. Characterization of the resistance genes is then carried out by direct sequencing. For this purpose primers for amplification or sequencing of the most representative aminoglycoside resistance genes were designed.

- Plasmid replicon typing. A methodology for the characterization of plasmid replicon types, as marker of plasmid backbone, was implemented to better understand the mechanisms of resistance spreading in the E. coli isolates from the ARS studies. This methodology is based on a multiplex-PCR (Carattoli et al., J Microbiol Methods 2005) and/or a DNA hybridization approach (Couturier et al., Microbiol Rev 1988). Several experiments were carried out to standardize the methodology in our laboratory, using a collection of plasmids harbouring known replicon types.

- Genotyping. A genotyping methodology was implemented to investigate the clonal relationships among the resistant E. coli isolates. This methodology is based on PCR amplification using three different decamer primers (Random Amplification of Polymorphic DNA, RAPD) (Pacheco et al., J Clin Microbiol 1997), and on a phylogenetic analysis carried out with the Diversity Database program (Bio-Rad Laboratories, Hercules, California) to compare different band patterns. Several experiments were carried out to standardize the methodology in our laboratory, using a collection of E. coli clinical isolates of different origins.